The extracellular matrix (ECM) represents an important morphological structure in which cells with their projections are organized in a complex tridimensional network with physical, chemical and electrical connections. In this network, cells produce proteases which play fundamental roles in the formation, maintenance and remodelling of tissues, in the transduction of inter- and intra-cellular signals and in cellular adhesion phenomena.
When the normal mechanism of control of network are lost in specific pathological conditions such as, for instance, in degenerated tumoral tissues, inflamUmatEed tissues, in the nervous, cardiac, vascular, osteoarticular, pulmonary, hepatic, renal tissues, in the skin, mucoses and glandular tissue, an hyperexpression of some zinc proteases belonging to various families is often observed. Among them, MMPs, ADAMs and ADAMTs play a fundamental role. In physiological conditions the normal tissue remodelling is under the control of two fundamental mechanisms that regulate the expression of MMPs, ADAMs and ADAMTs: the genic expression and/or the protein activation. The proteolytic activity of these zinc metalloproteinases is regulated at tissue level by several natural inhibitors, principally TIMPs (tissue inhibitor of metalloproteinases), which exert fundamental roles in the regulation of the activity of MMPs and also of the ADAMs and ADAMTs.
The loss of one of the mechanisms of control which are able to regulate the tissue levelof one of these zinc metalloproteinases in the ECM is often responsible for the development of serious degenerative processes which are characterized, beyond the alteration of the normal tissuemorphology, also by the presence and uncontrolled proliferation of cells of different type. The consequent morphological alteration leads to a tissue which has completely lost its normal characteristics and evolves in a non-physiological way.
Said alterations are typical both in highly aggressive tumoral forms and acute chronic inflammatory or immunologic pathologies. Therefore, high levels of these zinc metalloproteinases may be observed both in rapidly growing tumoral tissues and in metastasis, as well as in some pathologies of the central and peripheral nervous system, and of the cardiovascular, gastrointestinal, musculoskeletal, respiratory, renal, optical system, of the skin and of the mucosae. Diseases for which it has been demonstrated the certain pathological role related to the loss of control of the proteolytic activity of some zinc metalloproteinases include, for instance, highly metastatic tumoral forms, not solely tumoral angiogenesis, ictus and those forms with a varying degree of seriousness related to hypoxia and cerebral ischemia, atherosclerosis, Alzheimer's disease, coronary thrombosis, dilated cardiomyopathy, aortic aneurism, otosclerosis, proteinuria, multiple sclerosis, rheumatoid arthritis, osteoarthritis, septic arthritis, decubitus ulcers, pulmonary fibrosis, emphysema, periodontal disease as well as some infectious diseases.
Some of the zinc metalloproteinases present in the ECM are also involved in other biological processes such as ovulation, post-partum involution of uterus, breaking of the amyloid plaque at the level of the beta-amyloid precursor and inactivation of the protease alpha1 proteasic inhibitor (alpha1-PI). Therefore, the inhibition of such zinc metalloproteinases may represent a mechanism of control of fertility, as well as a mechanism able to increase the levels of alpha1-PI for the prevention of thological conditions such as emphysema and pulmonary, inflammatory and age-related diseases.
As long as about twenty years ago, it was thought that it was possible to use inhibitors of MMPs in a cytostatic therapeutic strategy in order to block tumours. Since then, two generations of MMPs inhibitors have been brought to clinical research, but only recently it has been foreseen the possibility that some inhibitors of MMPs might really be used in therapy [Abbenante Medicinal Chemistry, 2005, VOL 1, No. 1, 72]. The first generation inhibitors such as, for example, Ilonastat or Batimast, were highly effective but completely non-selective peptidomimetic inhibitors of MMPs. As such, they were unsuitable because they were not bioavailable through oral administration. A subsequent generation of inhibitors was developed, with molecules moderately selective towards the different MMPs; some of these inhibitors have reached phase III in clinical trial for the treatment of very aggressive forms of cancer. Nevertheless, in spite of remarkable efforts made with both classes, the research on the application of MMPs inhibitors in cancer therapy has brought discouraging results, as it was widely reported in some works [Zucker, S. Oncogene, 2000, 19, 6642; Coussens LM; Science, 2003, 295, 2387; Overall CM, et al Nat Rev Cancer, 2002, 2, 657; Fingleton B, Exp Opin Ther Thiget, 2003, 7, 385].
Nowadays only two molecules belonging to this pharmacological class are under development: Doxiciclina or Periostat, a well known tetracycline proposed for the therapy of periodontitis as inhibitor of MMP1/MMP2; and Glucosamina solfato, a well known drug today proposed as inhibitor of MMP8 in osteoarthritis.
One of the reasons for the lack of success of this pharmaceutical class of drugs, until now, has been the lack of high selectivity versus different MMPs.
At present, in fact, 24 different MMPs have been identified. Nevertheless, by using sophisticated techniques of genomics, proteomics and pharmacogenomics, only recently has it been possible to classify the different MMPs according to their single functions and physiological and pathological roles, therefore reclassifying them as therapeutic target or antitarget in the different types of pathologies. On the same basis, it has been possible to critically re-analyse some of the results obtained from the past trials. The reclassification of the various MMPs as target or antitarget has re-opened the field of research by making conceivable a third generation of inhibitors. On the basis of the knowledges acquired until now, it seems in fact possible to design drugs that might in fact be highly selective towards those MMPs which represent reliable targets for the degenerative pathologies of the extracellular matrix, such as the MMP2. More specifically, using MMP2 as the target, it is today possible to design and synthesize new effective inhibitors with high MMP1/MMP2 selectivity ratios, which show remarkable anti-invasive and anti-angiogenic properties in cellular models of highly aggressive tumours. On these basis we can today refer to MMPi target based therapy.
Also the ADAMs and the ADAMTs represent an important cellular target at the ECM level for the development of innovative drugs. These enzymes constitute a very large family: at least forty ADAMs have been characterised (twenty of them in the Homo sapiens species) and nearly ten ADAMTs have been isolated and sequenced from human species. These adamalysins, with their multiple physiological roles, contribute to the regulation of many cellular functions in tissues. These enzymes, strictly correlated to the more known MMPs, are zinc metalloproteinases themselves and almost always, they are anchored to the surface of the membranes exposed to the ECM and perform most of their proteolytic actions just in the ECM.
Their principal functions include the release and shedding of membrane proteins, of growth factors, of cytokines, the activation of receptors and the regulation of the cellular adhesion processes. As they have been isolated only recently, many of their actions are still unknown. Nevertheless, because of their functional and structural characteristics, they are correlated to the MMPs and some of them are inhibited by the same tissutal inhibitors which regulate the proteolytic action of MMPs, that it is to say the TIMPs. The hyperexpression of some of these adamalysins is related to the development of serious pathologies. The most known example is the ADAM17, (tumour necrosis factor alpha convertase), which normally activates the alpha-TNF and the receptors of various cytokines and also, if hyperexpressed, leads to an increase of TNF thus causing various pathologies among which are the rheumatoid arthritis, immunitary diseases, multiple sclerosis and cancer.
Also in this case, it is necessary to take advantage of the more recently acquired knowledge on the structural characteristics and the biological properties of the single adamalysins in order to design powerful and especially selective inhibitors to be proposed as potential drugs for the therapy of diseases such as, for instance, cancer, rheumatoid arthritis, ictus, atherosclerosis, multiple sclerosis, myocardial stroke and other cardiovascular diseases.
Efforts are thus devoted to extend also to the adamalysins the therapeutic approach of MMPi target based therapy previously illustrated, therefore avoiding problems and drawbacks known by the use of MMPs inhibitors which were non-selective for specific pathologies.
At present, several synthetic inhibitors of MMPs are known in the art [see, for example, Skiles J W Curr. Med. Chem. 2005, 11, 2911; Maskos, K Biochim. 2005, 87, 249; Fisher, J F et al Cancer Metastasis Rev. 2006, 25, 115]. Some of these inhibitors have been also studied and structurally modified to inhibit ADAMs [see, for example, Levin J I Curr. Top. Med. Chem. 2004, 4, 1289; Skiles J W Curr. Med. Chem. 2005, 11, 2911] and ADAMTs [see, for example, Yao, W et al J. Med. Chem. 2001, 44, 3347; WO01/87870; WO00/69839; Gavin, J. C. Curr. Pharm. Biotechn. 2006, 7, 25]. There exists, in addition, a large patent literature concerning zinc metalloproteinases inhibitors.
The most studied and described inhibitors present general structural characteristics commonly shared within the various classes: the presence of chelating groups for the catalytic zinc atom (ZBGs Zinc binding groups) which are able to give hydrogen bonds with the backbone of the target proteins and interactions of hydrophobic nature with the near pockets for the recognition of the substrate in S1, S1′, S2′ and in some cases S3′.
Numerous types of ZBGs have been studied so far, but the most widely known is the hydroxamic group which, giving two coordination bonds with zinc, leads to very powerful inhibitors in comparison with other chelating groups [see, as an example: WO 95/2982, WO 97/24117, WO 97/49679, EP 0780386, WO 90/05719, WO 93/20047, WO 06074, WO 00/46221, WO 00/44709, WO 99/25687, WO 00/50396, WO 00/69821, WO 04/071384, WO 99/41246, U.S. Pat. No. 6,750,228, US 2004/0127524, WO 2004/052840, WO 01/02369, WO 2004/000811, US 2003/0073845, and Fisher, J F et al Cancer Metastasis Rev. 2006, 25, 115]. Other chelating groups have been reported such as, for example, carboxylate, phosphonate, thiols [see, as an example: EP 1331224; EP 107736; WO 95/12389; WO 96/11209; U.S. Pat. No. 6,500,811; Fisher, J F et al Cancer Metastasis Rev. 2006, 25, 115; Breuer, E et al, Export Opin. Ther. Patents, 2005, 253] and many others [see, as an example: Puerta, D T et al J. Am. Chem. Soc, 2004, 126, 8388].
Very recent works in the study of the inhibitors of these zinc metalloproteinases concern the development of mechanism based inhibitors and the development of allosteric inhibitors [see, as an example: Kruger, A. et al Cancer Res. 2005, 65, 3523; US 2003/0129672, and US 2005/0004177].
Sulphonyl derivatives possessing MMPs inhibitory activity have been also disclosed in WO 00/69819 and WO 98/38859.
Among the compounds therein exemplified, some are characterized by the presence of a phenyl-sulphonyl-phenyl system bringing a carboxylate or hydroxamate group linked to a phenylene moiety, either directly or through an optionally substituted methylene group.
Today, it is clear that in order to develop a drug capable of inhibiting zinc metalloproteinases specifically produced in a determined degenerative pathology of the ECM, it is necessary to obtain highly selective inhibitors, targeted in a very specific manner against those specific metalloproteinases which are involved in the pathology of interest. For example, as pointed out before, it would be preferable to obtain high levels of selectivity of action towards MMP-2 and MMP-13 in case of potential inhibitors to be designed-for the anticancer therapy, with a contemporaneous low inhibition property against MMP-1, whose inhibition is known to cause musculoskeletal syndrome with fibroproliferative effects in the kneecaps [see, as an example: Hutchinson, J. W et al Bone Joint Surgery 1998, 80, 907; 18; Holmbeck, K. et al Cell 1999, 99, 81; Steward, W. P. Cancer Chemother. Pharmacol. 1999, 43, S56], against MMP-3, MMP-8 and MMP-9, which have been recently validated as antitarget for this kind of therapy [see, as an example: Overall CM, et al Nature Rew Cancer, 2006, 6, 227].
On the other hand, in the field of zinc metalloproteinases inhibitors to be used in degenerative non-tumoral pathologies, it would be advisable to obtain high levels of selectivity for MMP2, MMP8, MMP9, MMP13 and MMP14 [see, as example: Ishikawa, T et al Br. J. Pharm. 2005, 144, 133; Martel-Pelletier, J et al Best Pract. & Res. Clin. Rheum., 2001, 805-829] and for adamalysins as TACE (ADAM17) and ADAMTs-4 (aggrecanase-1) [see, as an example: US 2004/0110805; U.S. Pat. No. 5,770,624; WO 00/44709; US 2004/0110805; U.S. Pat. No. 6,500,811; Martel-Pelletier, J et al Best Pract. & Res. Clin. Rheum., 2001, 805-829].
Moreover, it would be preferable to replace the hydroxamate group (ZBG) with different clusters, less toxic, more stable in metabolism, more bioavailable and well absorbable via oral administration [see, as an example: Fisher, J F et al Cancer Metastasis Rev. 2006, 25, 115; Breuer, E et al Exp. Opin. Ther. Patents 2005, 15, 253; US 2005/6953788]Recently, the need to develop suitable diagnostic instruments for non-invasive technologies has been highlighted, such as PET (Positron Emission Tomography), SPECT (Single Photon Emission Tomography), MRI (Magnetic Resonance Imaging), OI (Optical Imaging) and Rx, able to find with high specificity degenerated tissues due to the hyperactivity of zinc metalloproteinases such as, for example, MMP2, MMP8, MMP9, MMP13, MMP14, TACE and ADAMTs1 and ADAMTs4[see, as example: Schafers, M. et al Basic
Science Rep. 2004, 11, 2554; Zheng, Qi-H et al Nucl. Med. Biol. 2002, 29, 761; Fei, X et al, Bioorg. & Med. Chem. Lett. 2003, 13, 2217; Zheng, Qi-H et al Nucl. Med. Biol. 2003, 30, 753; Kopta, K et al, Nucl. Med. Biol. 2004, 31, 257; Li, W P et al Q. J Nucl. Med. 2003, 47, 201; Haumber, R Curr. Pharm. Des. 2004, 10, 1439; WO 2005/049005].
Given the relevance of the inhibitors of zinc metalloproteinases in the prevention, treatment and/or diagnosis of pathological conditictns characterized by the absence of the normal tissue morphology and by the presence and uncontrolled proliferation of cells of different nature caused by loss of the physiological mechanisms of control in the ECM, and also considering the knowledge acquired in clinical trials on the known zinc metalloproteinases inhibitors, it would be very important to have new classes of molecules able to act with high selectivity and potency of action against some proteases specifically hyperexpressed in the various pathologies. In particular, it would be very important to have highly selective and powerful inhibitors for MMP2, MMP13 or MMP16, or for MMP2 and MMP14 (as for MMPs) or for TACE (for ADAMs) or again for ADAMTs1 and ADAMTs4 (for ADAMTs), which would be lacking as much as possible the inhibitory activity toward MMP1 and, according to the pathology, also toward MMP14.
At the same time, it would be highly desirable to have inhibitors less toxic, more metabolically stable, more bioavailable and well absorbable via oral administration, than the inhibitors known in the art.
These and other aims have been achieved by the thioaryl substituted zinc proteinase inhibitors of the present invention.
Said compounds are, in fact, able to inhibit the activation of specific MMPs (such as MMP2, MMP9, MMP13, MMP14 or MMP16) and/or ADAMs (such as TACE) and/or ADAMTs (such as ADAMTs1 or ADAMTs4) which are hyperexpressed in various degenerative diseases.
The compounds of the invention share a thioarylether type structure. This kind of structure allowg to modulation of the selectivity of the inhibitory action over various zinc proteases, according to the structural features of the compounds themeselves including: the oxidation state of the sulphur atom as sulphide, sulphoxide or sulphone (—S—, —SO—or —SO2—, respectively); the nature of the .zinc chelating group (ZBG) as well as the nature of the groups bound to it at P1, P1′ and P2′.
Generally, the compounds of the invention are inactive or scarcely active towards some MMPs such as MMP1, MMP3, MMP8 and MMP14, thus advantageously providing a reduction of therapeutic risk in relation to some tumoral pathologies in which these enzymes have been defined as antitarget [see, as example: Overall C M, et al Brit J of Cancer, 2006, 1; and Overall C M, et al Nature Rew Cancer, 2006, 6, 227].
In addition, thanks to their marked selectivity of action, the compounds of the invention are less cytotoxic than the inhibitors of the zinc proteinases known in the art.
As set forth below, the compounds of the invention may be thus used for preventive, therapeutic and cosmetic purposes and, also, as possible carriers of both diagnostic and therapeutically active agents, both in the field of medicine and veterinary medicine.